Nat. Chem. Biol. 1, 112–119 (2005); published online 29 May 2005; corrected after print 31 January 2013
In this Article1, we described a small-molecule inhibitor of necroptosis, termed Necrostatin-1 (Nec-1). Since the original publication, additional data regarding the properties of Nec-1 have been reported, including off-target activity and metabolic stability in mice, that are important in designing in vitro and, especially, in vivo experiments with Nec-1.
Teng et al.2 reported an optimized derivative of Nec-1, termed 7-Cl-O-Nec-1 (66 in ref. 2), that was used in ref. 1 to demonstrate the protection in an ischemic brain injury model. This molecule showed higher activity in inhibiting necroptosis in Jurkat cells than Nec-1 (EC50 = 210 nM versus EC50 = 490 nM), no nonspecific cytotoxicity at high concentrations (∼100 mM) and reasonable pharmacokinetic characteristics following intravenous administration in mice. Degterev et al.3 subsequently reported that Nec-1 shows limited metabolic stability, which is substantially improved with 7-Cl-O-Nec-1. Takahashi et al.4 also reported that Nec-1 showed paradoxical toxicity at lower, but not higher, doses in a mouse model of systemic inflammatory stress syndrome (SIRS). No such toxicity was observed with 7-Cl-O-Nec-1. Thus, for in-cell and in vivo experiments, we recommend the use of 7-Cl-O-Nec-1.
Muller et al.5 reported that Nec-1, also known by its chemical name of methylthiohydantoin-tryptophan, is a micromolar inhibitor of indolamine 2,3-deoxygenase (IDO) with EC50 = 11.4 mM in a cell-based assay. Thus, given the ∼20-fold higher activity of Nec-1 in a necroptotic assay, the use of lower concentrations of this molecule could be helpful in distinguishing between inhibition of necroptosis and IDO-related processes. Another known inhibitor of IDO, 1-methyl-DL-tryptophan, lacks activity against necroptosis as reported by both Degterev et al.3 and Takahashi et al.4 Notably, both reports show that optimized 7-Cl-O-Nec-1 lacks activity against IDO. Overall, potential nonspecific toxicity, inhibition of IDO and limited stability of Nec-1 should be taken into account when the molecule is used in vivo, whereas 7-Cl-O-Nec-1 lacks these liabilities and thus represents a superior choice for in vivo studies.
References
Degterev et al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat. Chem. Biol. 1, 112–119 (2005).
Teng et al. Structure-activity relationship study of novel necroptosis inhibitors. Bioorg. Med. Chem. Lett. 15, 5039–5044 (2005).
Degterev, A., Maki, J.L. & Yuan, J. Activity and specificity of necrostatin-1, small-molecule inhibitor of RIP1 kinase. Cell Death Differ. 20, 366 (2012)
Takahashi, N. et al. Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. Cell Death Dis. 3, e437 (2012)
Muller et al. Inhibition of indoleamine 2,3-dioxygenase, an immunoregulatory target of the cancer suppression gene Bin1, potentiates cancer chemotherapy. Nat. Med. 11, 312–319 (2005).
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Degterev, A., Huang, Z., Boyce, M. et al. Addendum: Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol 9, 192 (2013). https://doi.org/10.1038/nchembio0313-192a
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DOI: https://doi.org/10.1038/nchembio0313-192a